The herpes simplex virus type 1 (HSV-1) immediate-early protein ICP27 is essential for viral replication (56). ICP27 functions principally at the posttranscriptional level, affecting RNA processing and export (37,58,61). Early in infection, ICP27 associates with spliceosomal proteins (45,59,60) and mediates an inhibition of host cell splicing (3,19,34,62). This process contributes to the shutoff of host protein synthesis because cellular pre-mRNAs are incompletely spliced and thus are retained in the nucleus in stalled spliceosomal complexes. ICP27 inhibits host cell splicing by recruiting a primarily cytoplasmic kinase, termed SR protein kinase 1, to the nucleus, where its interaction with ICP27 alters its ability to phosphorylate essential splicing factors, termed SR proteins (62). This process results in stalled splicing complex formation (3,34,62). In metazoans, the nuclear export of mRNAs has been linked to pre-mRNA splicing (36,47,55). The basis of this connection was revealed by the discovery of a protein complex that is deposited on pre-mRNAs undergoing splicing at a specific position upstream of exon junctions (30)(31)(32)49). This exon junction complex (EJC) consists of at least six proteins, which have been shown to function in splicing, RNA export, cytoplasmic localization, mRNA surveillance, and translational efficiency (14,28,30,73). Here we show that these structures to which Aly/REF was redistributed colocalized with ICP4 and thus are sites of HSV-1 transcription. Further, ICP27 mutants that are unable to interact with Aly/REF were unable to recruit Aly/REF to centers of ICP4 staining; instead, Aly/REF remained associated with splicing factor SC35. However, a failure to interact with Aly/REF did not impair the export of ICP27 to the cytoplasm at late times after infection. Further, although it has been suggested that efficient shuttling of ICP27 requires RNA binding (67,68), an ICP27 mutant that lacks the essential RGG box RNA binding domain and thus cannot bind RNA (40, 58) was efficiently exported to the cytoplasm, whereas an ICP27 mutant that has a mutation in a predicted KH domain and that is able to bind RNA was largely retained in the nucleus.To further explore the export requirements for ICP27, we investigated its interaction with TAP/NXF1, the cellular mRNA export receptor. ICP27 was shown to interact
The host innate response to viral infection includes the production of interferons, which is dependent on the coordinated activity of multiple transcription factors. Herpes simplex virus 1 (HSV-1) has been shown to block efficient interferon expression by multiple mechanisms. We and others have demonstrated that HSV-1 can inhibit the transcription of genes promoted by interferon regulatory factor-3 (IRF-3), including interferon beta (IFN-beta), and that the immediate-early ICP0 protein is sufficient for this function. However, the exact mechanism by which ICP0 blocks IRF-3 activity has yet to be determined. Unlike some other viral proteins that inhibit IRF-3 activity, ICP0 does not appear to affect phosphorylation and dimerization of IRF-3. Here, we show that a portion of activated IRF-3 co-localizes with nuclear foci containing ICP0 at early times after virus infection. Co-localization to ICP0-containing foci is also seen with the IRF-3-binding partners and transcriptional co-activators, CBP and p300. In addition, using immunoprecipitation of infected cell lysates, we can immunoprecipitate a complex containing ICP0, IRF-3, and CBP. Thus we hypothesize that ICP0 recruits activated IRF-3 and CBP/p300 to nuclear structures, away from the host chromatin. This leads to the inactivation and accelerated degradation of IRF-3, resulting in reduced transcription of IFN-beta and an inhibition of the host response. Therefore, ICP0 provides an example of how viruses can block IFN-beta induction by sequestration of important transcription factors essential for the host response.
Posttranslational modification of histones is known to regulate chromatin structure and transcriptional activity, and the nuclear lamina is thought to serve as a site for heterochromatin maintenance and transcriptional silencing. In this report, we show that the nuclear lamina can also play a role in the downregulation of heterochromatin and in gene activation. Herpes simplex virus DNA initiates replication in replication compartments near the inner edge of the nucleus, and histones are excluded from these structures. To define the role of nuclear lamins in HSV replication, we examined HSV infection in wild-type and A-type lamin–deficient (Lmna −/−) murine embryonic fibroblasts (MEFs). In Lmna −/− cells, viral replication compartments are reduced in size and fail to target to the nuclear periphery, as observed in WT cells. Chromatin immunoprecipitation and immunofluorescence studies demonstrate that HSV DNA is associated with increased heterochromatin in Lmna −/− MEFs. These results argue for a functional role for A-type lamins as viral gene expression, DNA replication, and growth are reduced in Lmna −/− MEFs, with the greatest effect on viral replication at low multiplicity of infection. Thus, lamin A/C is required for targeting of the viral genome and the reduction of heterochromatin on viral promoters during lytic infection. The nuclear lamina can serve as a molecular scaffold for DNA genomes and the protein complexes that regulate both euchromatin and heterochromatin histone modifications.
Little is known about the mechanisms of gene targeting within the nucleus and its effect on gene expression, but most studies have concluded that genes located near the nuclear periphery are silenced by heterochromatin. In contrast, we found that early herpes simplex virus (HSV) genome complexes localize near the nuclear lamina and that this localization is associated with reduced heterochromatin on the viral genome and increased viral immediate-early (IE) gene transcription. In this study, we examined the mechanism of this effect and found that input virion transactivator protein, virion protein 16 (VP16), targets sites adjacent to the nuclear lamina and is required for targeting of the HSV genome to the nuclear lamina, exclusion of heterochromatin from viral replication compartments, and reduction of heterochromatin on the viral genome. Because cells infected with the VP16 mutant virus in1814 showed a phenotype similar to that of lamin A/C−/− cells infected with wild-type virus, we hypothesized that the nuclear lamina is required for VP16 activator complex formation. In lamin A/C−/− mouse embryo fibroblasts, VP16 and Oct-1 showed reduced association with the viral IE gene promoters, the levels of VP16 and HCF-1 stably associated with the nucleus were lower than in wild-type cells, and the association of VP16 with HCF-1 was also greatly reduced. These results show that the nuclear lamina is required for stable nuclear localization and formation of the VP16 activator complex and provide evidence for the nuclear lamina being the site of assembly of the VP16 activator complex.
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