The herpes simplex virus immediate-early ubiquitin ligase ICP0 induces degradation of the ICP0 repressor protein E2FBP1.

E2FBP1/hDRIL1, a DNA-binding A/T-rich interaction domain (ARID) family transcription factor, is expressed ubiquitously in human tissues and plays an essential role in maintaining the proliferation potential of passage-limited human fibroblasts by dissociating promyelocytic leukemia nuclear bodies (PML-NBs). This effect on PML-NBs is similar to that of viral immediate-early gene products, such as infected cellular protein 0 (ICP0) from human herpes simplex virus 1 (HSV-1), which also disrupts PML-NBs to override the intrinsic cellular defense. Here we report that E2FBP1 inhibits accumulation of ICP0 RNA and, at the same time, is degraded via ICP0's herpes ubiquitin ligase 2 (HUL-2) activity upon HSV-1 infection. These reciprocal regulatory roles of ICP0 and E2FBP1 are linked in an ARID-dependent fashion. Our results suggest that E2FBP1 functions as an intrinsic cellular defense factor in spite of its PML-NB dissociation function.

The RING finger domain of Varicella-Zoster virus ORF61p has E3 ubiquitin ligase activity that is essential for efficient autoubiquitination and dispersion of Sp100-containing nuclear bodies.

Varicella zoster virus encodes an immediate-early (IE) protein termed ORF61p that is orthologous to the herpes simplex virus IE protein ICP0. Although these proteins share several functional properties, ORF61p does not fully substitute for ICP0. The greatest region of similarity between these proteins is a RING finger domain. We demonstrate that disruption of the ORF61p RING finger domain by amino acid substitution (Cys19Gly) alters ORF61p intranuclear distribution and abolishes ORF61p-mediated dispersion of Sp100-containing nuclear bodies. In addition, we demonstrate that an intact ORF61p RING finger domain is necessary for E3 ubiquitin ligase activity and is required for autoubiquitination and regulation of protein stability.

The co-chaperone BAG3 regulates Herpes Simplex Virus replication.

Viruses as obligate intracellular parasites use host cell proteins to ensure efficient replication and spread. Cellular proteins are required for several stages of a virus life cycle. Here, we identify BAG3, a co-chaperone, as a regulator of herpes virus immediate early gene expression. We report that a herpes simplex virus lacking the gene encoding a potent transcriptional activator, ICP0, is compromised for replication in cells silenced for BAG3 in a multiplicity of infection-dependent manner. We also show a requirement for BAG3 to augment virus gene expression and demonstrate that the co-chaperone acts independently of promyelocytic leukemia to increase herpes simplex virus replication.

Identification of phosphorylated residues on varicella-zoster virus immediate-early protein ORF63.

Efficient replication of varicella-zoster virus (VZV) in cell culture requires expression of protein encoded by VZV open reading frame 63 (ORF63p). Two-dimensional gel analysis demonstrates that ORF63p is extensively modified. Mass spectroscopy analysis of ORF63p isolated from transiently transfected HEK 293 and stably transfected MeWo cells identified 10 phosphorylated residues. In VZV-infected MeWo cells, only six phosphorylated residues were detected. This report identifies phosphorylation of two previously uncharacterized residues (Ser5 and Ser31) in ORF63p extracted from cells infected with VZV or transfected with an ORF63p expression plasmid. Computational analysis of ORF63p for known kinase substrates did not identify Ser5 or Ser31 as candidate phosphorylation sites, suggesting that either atypical recognition sequences or novel cellular kinases are involved in ORF63p post-translational modification.

Components of nuclear domain 10 bodies regulate varicella-zoster virus replication.

PML, Sp100, and Daxx are proteins that normally reside within nuclear domains 10 (ND10s). They associate with DNA virus genomes and repress the very early stages of the DNA virus replication cycle. Virus-encoded proteins counteract this innate antiviral response. ICP0, a herpes simplex virus (HSV) immediate-early protein, is necessary and sufficient to dissociate ND10s and target their two major components, PML and Sp100, for proteasomal degradation. In this report, we show that ORF61p, the varicella-zoster virus (VZV) ortholog of ICP0, does not degrade PML and alters Sp100 levels only slightly. Furthermore, we demonstrate that other virus proteins cannot substitute for this lack of function during infection. By using short interfering RNAs, we depleted PML, Sp100, and Daxx and studied their roles in plaquing efficiency, virus protein accumulation, infectious-center titer, and virus spread. The results of these studies show that components of ND10s can accelerate VZV replication but do not ultimately control cell-associated virus titers. We conclude that while both ICP0 and ORF61p activate virus gene expression, they modulate host innate repression mechanisms in two different ways. As a result, HSV and VZV commandeer their host cells by distinct mechanisms to ensure their replication and spread.

Complementation of a herpes simplex virus ICP0 null mutant by varicella-zoster virus ORF61p.

The herpes simplex virus (HSV) ICP0 protein acts to overcome intrinsic cellular defenses that repress viral alpha gene expression. In that vein, viruses that have mutations in ICP0's RING finger or are deleted for the gene are sensitive to interferon, as they fail to direct degradation of promyelocytic leukemia protein (PML), a component of host nuclear domain 10s. While varicella-zoster virus is also insensitive to interferon, ORF61p, its ICP0 ortholog, failed to degrade PML. A recombinant virus with each coding region of the gene for ICP0 replaced with sequences encoding ORF61p was constructed. This virus was compared to an ICP0 deletion mutant and wild-type HSV. The recombinant degraded only Sp100 and not PML and grew to higher titers than its ICP0 null parental virus, but it was sensitive to interferon, like the virus from which it was derived. This analysis permitted us to compare the activities of ICP0 and ORF61p in identical backgrounds and revealed distinct biologic roles for these proteins.

Chaperone-fusion expression plasmid vectors for improved solubility of recombinant proteins in Escherichia coli.

The enteric bacterium Escherichia coli is the most extensively used prokaryotic organism for production of proteins of therapeutic or commercial interest. However, it is common that heterologous over-expressed recombinant proteins fail to properly fold resulting in formation of insoluble aggregates known as inclusion bodies. Complex systems have been developed that employ simultaneous over-expression of chaperone proteins to aid proper folding and solubility during bacterial expression. Here we describe a simple method whereby a protein of interest, when fused in frame to the E. coli chaperones DnaK or GroEL, is readily expressed in large amounts in a soluble form. This system was tested using expression of the mouse prion protein PrP, which is normally insoluble in bacteria. We show that while in trans over-expression of the chaperone DnaK failed to alter partitioning of PrP from the insoluble inclusion body fraction to the soluble cytosol, expression of a DnaK-PrP fusion protein yielded large amounts of soluble protein. Similar results were achieved with a fragment of insoluble Varicella Zoster virus protein ORF21p. In theory this approach could be applied to any protein that partitions with inclusion bodies to render it soluble for production in E. coli.

PATJ, a tight junction-associated PDZ protein, is a novel degradation target of high-risk human papillomavirus E6 and the alternatively spliced isoform 18 E6.

The E6 protein from high-risk human papillomavirus types interacts with and degrades several PDZ domain-containing proteins that localize to adherens junctions or tight junctions in polarized epithelial cells. We have identified the tight junction-associated multi-PDZ protein PATJ (PALS1-associated TJ protein) as a novel binding partner and degradation target of high-risk types 16 and 18 E6. PATJ functions in the assembly of the evolutionarily conserved CRB-PALS1-PATJ and Par6-aPKC-Par3 complexes and is critical for the formation of tight junctions in polarized cells. The ability of type 18 E6 full-length to bind to, and the subsequent degradation of, PATJ is dependent on its C-terminal PDZ binding motif. We demonstrate that the spliced 18 E6* protein, which lacks a C-terminal PDZ binding motif, associates with and degrades PATJ independently of full-length 18 E6. Thus, PATJ is the first binding partner that is degraded in response to both isoforms of 18 E6. The ability of E6 to utilize a non-E6AP ubiquitin ligase for the degradation of several PDZ binding partners has been suggested. We also demonstrate that 18 E6-mediated degradation of PATJ is not inhibited in cells where E6AP is silenced by shRNA. This suggests that the E6-E6AP complex is not required for the degradation of this protein target.

BAG3, a host cochaperone, facilitates varicella-zoster virus replication.

Varicella-zoster virus (VZV) establishes a lifelong latent infection in the dorsal root ganglia of the host. During latency, a subset of virus-encoded regulatory proteins is detected; however, they are excluded from the nucleus. ORF29p, a single-stranded DNA binding protein, is one of these latency-associated proteins. We searched for cell proteins that interact with ORF29p and identified BAG3. BAG3, Hsp70/Hsc70, and Hsp90 colocalize with ORF29p in nuclear transcription/replication factories during lytic replication of VZV. Pharmacological intercession of Hsp90 activity with ansamycin antibiotics or depletion of BAG3 by small interfering RNA results in inhibition of virus replication. Replication in BAG3-depleted cell lines is restored by complementation with exogenous BAG3. Alteration of host chaperone activity provides a novel means of regulating virus replication.

Posttranslational modification and cell type-specific degradation of varicella-zoster virus ORF29p.

The ORF29 gene of varicella-zoster virus encodes a single-stranded DNA binding protein that is predominantly nuclear during lytic infection but appears to be restricted to the cytoplasm of latently infected neurons. Following reactivation, ORF29p accumulates in the nuclei of neurons, suggesting that its confinement to the cytosol may be critical for maintaining quiescence. When autonomously expressed, ORF29p accumulates in the nuclei of fibroblasts and the cytoplasm of cells (guinea pig enteric neurons) and cell lines (U373MG) of neuronal origin. Inhibition of the 26S proteasome redirects the accumulation of ORF29p to the nucleus in cells of neuronal origin. Here, we show that ORF29p is ubiquitinated and sumoylated in 293T cells and subsequently degraded from the N terminus. Ubiquitinated ORF29p accumulates in both the nuclei and the cytoplasm of fibroblasts, but degradation products are seen primarily in the cytoplasm. Modification and degradation of ORF29p occurs in 293T, U373MG, and MeWo cells. Therefore, these processes are ubiquitous; however, the robustness of the degradation process is cell type specific. The proteasome-mediated mechanism of nuclear exclusion in U373MG cells is an active process that is not specific for the endogenous ORF29p nuclear localization signal but can be saturated by protein stabilization or overexpression, which leads to nuclear accumulation of ORF29p. The evidence for ORF29p ubiquitination and previous data regarding the effect of proteasome inhibitors on the abundance and distribution of ORF29p implicate the 26S proteasome in influencing the protein's cell type-specific localization.

UL54-null pseudorabies virus is attenuated in mice but productively infects cells in culture.

The pseudorabies virus (PRV) UL54 homologs are important multifunctional proteins with roles in shutoff of host protein synthesis, transactivation of virus and cellular genes, and regulation of splicing and translation. Here we describe the first genetic characterization of UL54. We constructed UL54 null mutations in a PRV bacterial artificial chromosome using sugar suicide and lambdaRed allele exchange systems. Surprisingly, UL54 is dispensable for growth in tissue culture but exhibits a small-plaque phenotype that can be complemented in trans by both the herpes simplex virus type 1 ICP27 and varicella-zoster virus open reading frame 4 proteins. Deletion of UL54 in the virus vJSdelta54 had no effect on the ability of the virus to shut off host cell protein synthesis but did affect virus gene expression. The glycoprotein gC accumulated to lower levels in cells infected with vJSdelta54 compared to those infected with wild-type virus, while gK levels were undetectable. Other late gene products, gB, gE, and Us9, accumulated to higher levels than those seen in cells infected with wild-type virus in a multiplicity-dependent manner. DNA replication is also reduced in cells infected with vJSdelta54. UL54 appears to regulate UL53 and UL52 at the transcriptional level as their respective RNAs are decreased in cells infected with vJSdelta54. Interestingly, vJSdelta54 is highly attenuated in a mouse model of PRV infection. Animals infected with vJSdelta54 survive twice as long as animals infected with wild-type virus, and this results in delayed accumulation of virus-specific antigens in skin, dorsal root ganglia, and spinal cord tissues.

The cellular localization pattern of Varicella-Zoster virus ORF29p is influenced by proteasome-mediated degradation.

Varicella-zoster virus (VZV) open reading frame 29 (ORF29) encodes a single-stranded DNA binding protein. During lytic infection, ORF29p is localized primarily to infected-cell nuclei, whereas during latency it appears in the cytoplasm of infected neurons. Following reactivation, ORF29p accumulates in the nucleus. In this report, we analyze the cellular localization patterns of ORF29p during VZV infection and during autonomous expression. Our results demonstrate that ORF29p is excluded from the nucleus in a cell-type-specific manner and that its cellular localization pattern may be altered by subsequent expression of VZV ORF61p or herpes simplex virus type 1 ICP0. In these cases, ORF61p and ICP0 induce nuclear accumulation of ORF29p in cell lines where it normally remains cytoplasmic. One cellular system utilized by ICP0 to influence protein abundance is the proteasome degradation pathway. Inhibition of the 26S proteasome, but not heat shock treatment, resulted in accumulation of ORF29p in the nucleus, similar to the effect of ICP0 expression. Immunofluorescence microscopy and pulse-chase experiments reveal that stabilization of ORF29p correlates with its nuclear accumulation and is dependent on a functional nuclear localization signal. ORF29p nuclear translocation in cultured enteric neurons and cells derived from an astrocytoma is reversible, as the protein's distribution and stability revert to the previous states when the proteasomal activity is restored. Thus, stabilization of ORF29p leads to its nuclear accumulation. Although proteasome inhibition induces ORF29p nuclear accumulation, this is not sufficient to reactivate latent VZV or target the immediate-early protein ORF62p to the nucleus in cultured guinea pig enteric neurons.

An early regulatory function required in a cell type-dependent manner is expressed by the genomic but not the cDNA copy of the herpes simplex virus 1 gene encoding infected cell protein 0.

The alpha 0 genes of herpes simplex virus 1 (HSV-1) contain three exons. Earlier studies have shown that the substitution of genomic sequences with a cDNA copy does not alter the capacity of the virus to replicate or establish latent infection. Other studies have demonstrated that HSV-1 may express alternatively spliced forms of alpha 0 transcripts. The studies reported here centered on a mutant HSV-1(vCPc0) strain in which the genomic copies of the alpha 0 gene were replaced with cDNA copies. From our research, we report the following observations. (i) In contrast to events transpiring in cells infected with wild-type virus, the expression of HSV-1(vCPc0) genes was delayed or restricted to alpha genes for many hours in rabbit skin cells and to a lesser extent in HEp-2 cells but not in Vero cells. This delay in the expression of HSV-1(vCPc0) beta or gamma genes was also multiplicity of infection dependent. (ii) Exposure to MG132, a proteasomal inhibitor, before infection with wild-type virus had no significant effect on the accumulation of viral proteins in Vero cells and caused an only slight delay in viral gene expression in rabbit skin cells in a multiplicity of infection-dependent fashion. The drug had no effect when it was added to the medium 3 h after infection. (iii) Rabbit skin or HEp-2 cells exposed to MG132 3 h after infection with the HSV-1(vCPc0) mutant accumulated only alpha proteins. This restriction was cell type dependent but not multiplicity of infection dependent. (iv) Both the delay in the expression of beta and gamma genes and the effect of MG132 added to the medium 3 h after infection were rescued by restoration of the intron 1 sequences in the HSV-1(vCPc0) mutant. However, cells transduced by baculoviruses expressing intron 1 RNA did not complement the HSV-1(vCPc0) mutant, suggesting that the function of intron 1 is in cis rather than in trans. We came to the following conclusions as a result. (i) Post-alpha gene expression requires the involvement of the proteasomal pathway in a cell type-dependent manner. Consistent with this requirement, the proapoptotic functions of MG132 are blocked in cells infected before exposure to the drug but not after exposure. (ii) A function encoded by the alpha 0 gene that is absent from the cDNA copy is required for viral gene expression in a cell type- and multiplicity of infection-dependent fashion. The absence of this master function delays but does not ultimately block viral gene expression in the cell lines tested here.

The Epstein-Barr virus SM protein is functionally similar to ICP27 from herpes simplex virus in viral infections.

The herpes simplex virus type 1 (HSV-1) ICP27 protein is an essential RNA-binding protein that shuttles between the nucleus and cytoplasm to increase the cytoplasmic accumulation of viral late mRNAs. ICP27 homologs have been identified in each of the herpesvirus subfamilies, and accumulating evidence indicates that homologs from the gammaherpesvirus subfamily function similarly to ICP27. In particular, the Epstein-Barr virus (EBV) SM protein posttranscriptionally regulates gene expression, binds RNA in vitro and in vivo, and shuttles between the nucleus and cytoplasm. To determine if these two proteins function through a common mechanism, the ability of EBV SM to complement the growth defect of an HSV-1 ICP27-null virus was examined in a transient-expression assay. ICP27 stimulated the growth of the null mutant more efficiently than did SM, but the ability of SM to compensate for the ICP27 defects suggests conservation of common functions. To assay for complementation in the context of a viral infection, the growth properties of an HSV recombinant expressing SM in an ICP27-null background were analyzed. SM stimulated growth of the recombinant, although this growth was reduced by comparison to that of an ICP27-expressing virus. By contrast, an HSV recombinant expressing an SM mutant allele defective for transactivation activity and nucleocytoplasmic shuttling did not grow at all. These results suggest that SM and ICP27 may regulate gene expression through a common pathway that is evolutionarily conserved in herpesviruses.