Stephen A. Rice

PiggyBac Transposon Mutagenesis: A Tool for Cancer Gene Discovery in Mice

Piggybacking on Cancer Genes Transposons are mobile segments of DNA that can insert in or near important genes to cause mutations that disrupt gene function. Rad et al. (p. 1104, published online 14 October) adapted a mutagenic transposon called Piggybac, originally derived from a moth, into a tool for discovery of cancer-causing genes in mice. Mobilization of Piggybac in mice was associated with the development of leukemias and solid tumors. In many instances the causative mutations, which were identified by mapping the Piggybac integration sites, were within genes not previously implicated in cancer. Mutations induced by a transposable element in mice can be used to identify cancer-causing genes. Transposons are mobile DNA segments that can disrupt gene function by inserting in or near genes. Here, we show that insertional mutagenesis by the PiggyBac transposon can be used for cancer gene discovery in mice. PiggyBac transposition in genetically engineered transposon-transposase mice induced cancers whose type (hematopoietic versus solid) and latency were dependent on the regulatory elements introduced into transposons. Analysis of 63 hematopoietic tumors revealed that PiggyBac is capable of genome-wide mutagenesis. The PiggyBac screen uncovered many cancer genes not identified in previous retroviral or Sleeping Beauty transposon screens, including Spic, which encodes a PU.1-related transcription factor, and Hdac7, a histone deacetylase gene. PiggyBac and Sleeping Beauty have different integration preferences. To maximize the utility of the tool, we engineered 21 mouse lines to be compatible with both transposon systems in constitutive, tissue- or temporal-specific mutagenesis. Mice with different transposon types, copy numbers, and chromosomal locations support wide applicability.

Accumulation of Herpes Simplex Virus Type 1 Early and Leaky-Late Proteins Correlates with Apoptosis Prevention in Infected Human HEp-2 Cells

ABSTRACT We previously reported that a recombinant ICP27-null virus stimulated, but did not prevent, apoptosis in human HEp-2 cells during infection (M. Aubert and J. A. Blaho, J. Virol. 73:2803–2813, 1999). In the present study, we used a panel of 15 recombinant ICP27 mutant viruses to determine which features of herpes simplex virus type 1 (HSV-1) replication are required for the apoptosis-inhibitory activity. Each virus was defined experimentally as either apoptotic, partially apoptotic, or nonapoptotic based on infected HEp-2 cell morphologies, percentages of infected cells with condensed chromatin, and patterns of specific cellular death factor processing. Viruses d27-1, d1-5,d1-2, M11, M15, M16, n504R,n406R, n263R, and n59R are apoptotic or partially apoptotic in HEp-2 cells and severely defective for growth in Vero cells. Viruses d2-3,d3-4, d4-5, d5-6, andd6-7 are nonapoptotic, demonstrating that ICP27 contains a large amino-terminal region, including its RGG box RNA binding domain, which is not essential for apoptosis prevention. Accumulations of viral TK, VP16, and gD but not gC, ICP22, or ICP4 proteins correlated with prevention of apoptosis during the replication of these viruses. Of the nonapoptotic viruses, d4-5 did not produce gC, indicating that accumulation of true late gene products is not necessary for the prevention process. Analyses of viral DNA synthesis in HEp-2 cells indicated that apoptosis prevention by HSV-1 requires that the infection proceeds to the stage in which viral DNA replication takes place. Infections performed in the presence of the drug phosphonoacetic acid confirmed that the process of viral DNA synthesis and the accumulation of true late (γ2) proteins are not required for apoptosis prevention. Based on our results, we conclude that the accumulation of HSV-1 early (β) and leaky-late (γ1) proteins correlates with the prevention of apoptosis in infected HEp-2 cells.

Herpes Simplex Virus Immediate-Early Protein ICP22 Triggers Loss of Serine 2-Phosphorylated RNA Polymerase II

ABSTRACT During eukaryotic mRNA transcription, the synthetic activity and mRNA processing factor interactions of RNA polymerase II (RNAP II) are regulated by phosphorylation of its carboxyl-terminal domain (CTD), with modification occurring primarily on serines 2 and 5 of the CTD. We previously showed that herpes simplex virus type 1 (HSV-1) infection rapidly triggers the loss of RNAP II forms bearing serine 2 phosphorylation (Ser-2P RNAP II). Here we show that the HSV-1 immediate-early (IE) protein ICP22 is responsible for this effect during the IE phase of infection. This activity does not require the viral UL13 protein kinase, which is required for several other regulatory functions of ICP22. Additionally, we show that transient expression of ICP22 can trigger the loss of Ser-2P RNAP II in transfected cells. Thus, the ability of ICP22 to cause the loss of Ser-2 RNAP II does not require other viral factors or the context of the infected cell. Expression of the HSV-1 ICP22-related protein US1.5, which corresponds to residues 147 to 420 of ICP22, also triggers a loss of Ser-2P RNAP II in transfected cells, whereas expression of the varicella-zoster virus ICP22 homolog, ORF63, does not. Our study also provides evidence for a second, viral late gene-dependent pathway that triggers loss of Ser-2P RNAP II in infected cells, consistent with the recent work of Dai-Ju et al. (J. Q. Dai-Ju, L. Li, L. A. Johnson, and R. M. Sandri-Goldin, J. Virol. 80:3567-3581, 2006). Therefore, it appears that HSV-1 has evolved redundant mechanisms for triggering the loss of a specific phosphorylated form of RNAP II.

Mapping of Functional Regions in the Amino-Terminal Portion of the Herpes Simplex Virus ICP27 Regulatory Protein: Importance of the Leucine-Rich Nuclear Export Signal and RGG Box RNA-Binding Domain

ABSTRACT Infected-cell protein 27 (ICP27) is an essential herpes simplex virus type 1 (HSV-1) regulatory protein that activates a subset of viral delayed-early and late genes, at least in part through posttranscriptional mechanisms. Previous studies have shown that the amino (N)-terminal half of the protein contains important functional regions, including a leucine-rich nuclear export signal (NES). However, to date, the phenotype of an HSV-1 ICP27 NES mutant has not been reported. In this study, we engineered and characterized dLeu, an HSV-1 deletion mutant that specifically lacks ICP27s NES (amino acids 6 to 19). The phenotype of dLeu was analyzed alongside those of eight other ICP27 N-terminal deletion mutants. We found that in Vero cells, dLeu displays modest defects in viral gene expression and an approximately 100-fold reduction in the production of viral progeny. Unlike wild-type (WT) ICP27, which exhibits a cytoplasmic distribution in addition to its predominant nuclear localization, dLeu ICP27 is highly restricted to the cell nucleus. This strongly suggests that the N-terminal leucine-rich sequence functions as an NES during viral infection. Our analysis of dLeu and the other mutants has enabled us to genetically define the regions in the N-terminal 200 residues of ICP27 which are required for efficient viral growth in Vero cells. Only two regions appear to be important: (i) the leucine-rich NES and (ii) the RGG box RNA-binding domain, encoded by residues 139 to 153. A virus lacking the RGG box-encoding sequence, d4-5, has a phenotype similar to that of dLeu in that it displays modest defects in viral gene expression and grows poorly. Interestingly, deletion of both the NES and RGG box, as well as the sequences in between, is lethal. The resulting virus, d1-5, displays severe defects in viral gene expression and DNA synthesis and is unable to produce significant amounts of infectious progeny. Therefore, the N-terminal portion of ICP27 contains at least two functional domains which collectively are absolutely essential for viral infection.

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

ABSTRACT 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.

Herpes Simplex Virus Type 1 ICP27-Dependent Activation of NF-κB

ABSTRACT The ability of herpes simplex virus type 1 (HSV-1) to activate NF-κB has been well documented. Beginning at 3 to 5 h postinfection, HSV-1 induces a robust and persistent nuclear translocation of an NF-κB-dependent (p50/p65 heterodimer) DNA binding activity, as measured by electrophoretic mobility shift assay. Activation requires virus binding and entry, as well as de novo infected-cell protein synthesis, and is accompanied by loss of both IκBα and IκBβ. In this study, we identified loss of IκBα as a marker of NF-κB activation, and infection with mutants with individual immediate-early (IE) regulatory proteins deleted indicated that ICP27 was necessary for IκBα loss. Analysis of both N-terminal and C-terminal mutants of ICP27 identified the region from amino acids 21 to 63 as being necessary for IκBα loss. Additional experiments with mutant viruses with combinations of IE genes deleted revealed that the ICP27-dependent mechanism of NF-κB activation may be augmented by functional ICP4. We also analyzed two additional markers for NF-κB activation, phosphorylation of the p65 subunit on Ser276 and Ser536. Phosphorylation of both serines was induced upon HSV infection and required functional ICP4 and ICP27. Pharmacological inhibitor studies revealed that both IκBα and Ser276 phosphorylation were dependent on Jun N-terminal protein kinase activity, while Ser536 phosphorylation was not affected during inhibitor treatment. These results demonstrate that there are several layers of regulation of NF-κB activation during HSV infection, highlighting the important role that NF-κB may play in infection.

CK2 Protein Kinase Is Stimulated and Redistributed by Functional Herpes Simplex Virus ICP27 Protein

ABSTRACT It has been shown previously (S. Wadd, H. Bryant, O. Filhol, J. E. Scott, T.-T. Hsieh, R. D. Everett, and J. B. Clements, J. Biol. Chem. 274:28991-28998, 2000) that ICP27, an essential and multifunctional herpes simplex virus type 1 (HSV-1) protein, interacts with CK2 and with heterogeneous ribonucleoprotein K (hnRNP K). CK2 is a pleiotropic and ubiquitous protein kinase, and the tetrameric holoenzyme consists of two catalytic α or α′ subunits and two regulatory β subunits. We show here that HSV-1 infection stimulates CK2 activity. CK2 stimulation occurs at early times after infection and correlates with redistribution of the holoenzyme from the nucleus to the cytoplasm. Both CK2 stimulation and redistribution require expression and cytoplasmic accumulation of ICP27. In HSV-1-infected cells, CK2 phosphorylates ICP27 and affects its cytoplasmic accumulation while it also phosphorylates hnRNP K, which is not ordinarily phosphorylated by this kinase, suggesting an alteration of hnRNP K activities. This is the first example of CK2 stimulation by a viral protein in vivo, and we propose that it might facilitate the HSV-1 lytic cycle by, for example, regulating trafficking of ICP27 protein and/or viral RNAs.

Herpes Simplex Virus Type 1 Immediate-Early Protein ICP27 Is Required for Efficient Incorporation of ICP0 and ICP4 into Virions

ABSTRACT Early in infection, herpes simplex virus type 1 (HSV-1) immediate-early (IE) proteins ICP0 and ICP4 localize to the nucleus, where they stimulate viral transcription. Later in infection, ICP0 and to a lesser extent ICP4 accumulate in the cytoplasm, but their biological role there is unknown. Previously, it was shown that the cytoplasmic localization of ICP0/4 requires the multifunctional IE protein ICP27, which is itself an activator of viral gene expression. Here, we identify a viral ICP27 mutant, d3-4, which is unable to efficiently localize ICP0 and ICP4 to the cytoplasm but which otherwise resembles wild-type HSV-1 in its growth and viral gene expression phenotypes. These results genetically separate the function of ICP27 that affects ICP0/4 localization from its other functions, which affect viral growth and gene expression. As both ICP0 and ICP4 are known to be minor virion components, we used d3-4 to test the hypothesis that the cytoplasmic localization of these proteins is required for their incorporation into viral particles. Consistent with this conjecture, d3-4 virions were found to lack ICP0 in their tegument and to have greatly reduced levels of ICP4. Thus, the cytoplasmic localization of ICP0 and ICP4 appears to be a prerequisite for the assembly of these important transcriptional regulatory proteins into viral particles. Furthermore, our results show that ICP27 plays a previously unrecognized role in determining the composition of HSV-1 virions.

ICP22 is required for wild-type composition and infectivity of herpes simplex virus type 1 virions

ABSTRACT The immediate-early regulatory protein ICP22 is required for efficient replication of herpes simplex virus type 1 in some cell types (permissive) but not in others (restrictive). In mice infected via the ocular route, the pathogenesis of an ICP22− virus, 22/n199, was altered relative to that of wild-type virus. Specifically, tear film titers of 22/n199-infected mice were significantly reduced at 3 h postinfection relative to those of mice infected with wild-type virus. Further, 22/n199 virus titers were below the level of detection in trigeminal ganglia (TG) during the first 9 days postinfection. On day 30 postinfection, TG from 22/n199-infected mice contained reduced viral genome loads and exhibited reduced expression of latency-associated transcripts and reduced reactivation efficiency relative to TG from wild-type virus-infected mice. Notably, the first detectable alteration in the pathogenesis of 22/n199 in these tests occurred in the eye prior to the onset of nascent virus production. Thus, ICP22− virions appeared to be degraded, cleared, or adsorbed more rapidly than wild-type virions, implying potential differences in the composition of the two virion types. Analysis of the protein composition of purified extracellular virions indicated that ICP22 is not a virion component and that 22/n199 virions sediment at a reduced density relative to wild-type virions. Although similar to wild-type virions morphologically, 22/n199 virions contain reduced amounts of two γ2 late proteins, US11 and gC, and increased amounts of two immediate-early proteins, ICP0 and ICP4, as well as protein species not detected in wild-type virions. Although ICP22− viruses replicate to near-wild-type levels in permissive cells, the virions produced in these cells are biochemically and physically different from wild-type virions. These virion-specific differences in ICP22− viruses add a new level of complexity to the functional analysis of this immediate-early viral regulatory protein.

Herpes Simplex Virus Type 1 Immediate-Early Protein ICP22 Is Required for VICE Domain Formation during Productive Viral Infection

ABSTRACT During productive infection, herpes simplex virus type 1 (HSV-1) induces the formation of discrete nuclear foci containing cellular chaperone proteins, proteasomal components, and ubiquitinated proteins. These structures are known as VICE domains and are hypothesized to play an important role in protein turnover and nuclear remodeling in HSV-1-infected cells. Here we show that VICE domain formation in Vero and other cells requires the HSV-1 immediate-early protein ICP22. Since ICP22 null mutants replicate efficiently in Vero cells despite being unable to induce VICE domain formation, it can be concluded that VICE domain formation is not essential for HSV-1 productive infection. However, our findings do not exclude the possibility that VICE domain formation is required for viral replication in cells that are nonpermissive for ICP22 mutants. Our studies also show that ICP22 itself localizes to VICE domains, suggesting that it could play a role in forming these structures. Consistent with this, we found that ICP22 expression in transfected cells is sufficient to reorganize the VICE domain component Hsc70 into nuclear inclusion bodies that resemble VICE domains. An N-terminal segment of ICP22, corresponding to residues 1 to 146, is critical for VICE domain formation in infected cells and Hsc70 reorganization in transfected cells. We previously found that this portion of the protein is dispensable for ICP22s effects on RNA polymerase II phosphorylation. Thus, ICP22 mediates two distinct regulatory activities that both modify important components of the host cell nucleus.